Project Summary Apolipoprotein (apo) A-I is a multifunctional protein with a well-established role in reverse cholesterol transport and is an important player in heart disease. It is the main protein component of high-density lipoprotein (HDL), which circulates through plasma promoting cholesterol efflux. While a high-resolution structure is not known yet, extensive biophysical analysis has suggested that the 28 kDa protein is made of two domains, each of which contain amphipathic ?-helices for association with lipid surfaces. The C-terminal (CT) domain contains helical segments that initiate lipid binding, and is also the site responsible for self-association. It is a critical part of the protein needed for maturation of lipid-free apoA-I into HDL. Conflicting data exist about the role of the N-terminal (NT) helices in this process, as well as the precise helical segments of the CT domain. We have recently discovered that CT lysine residues are critical for self-association, and were able to create a monomeric version of the protein. To identify apoA-I helical segments important for initiation of lipid binding and self-association, which are closely connected, we developed a chimeric protein. This chimera will be used to identify which helical segments of apoA-I, both NT and CT ?-helices, are required for initiation of lipid binding and self-association. To identify the specific amino acid residues of the CT domain required in self-association, site-directed mutagenesis will be employed. All proteins will be expressed in a bacterial expression system, purified by affinity and size-exclusion chromatography, and characterized for structure and function. The results of this study will lead to a much better understanding in the domain organization of this critical apolipoprotein, their structure function relationship, and may also provide opportunities for high-resolution structural analysis using monomeric apoA-I.